Recently, various methods have been developed for delivering macromolecules such as therapeutic drug, peptides and proteins into cells in vitro and in vivo.
In vitro methods include electroporation, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium-phosphate-DNA precipitate, DEAE-dextran mediated transfection, infection with modified viral nucleic acids, and direct micro-injection into single cells. But such methods are of extremely limited usefulness for delivery of proteins.
Delivery of macromolecules into cells in vivo has been accomplished with scrape loading, calcium phosphate precipitates and liposomes. However, these techniques have, up to date, shown limited usefulness for in vivo cellular delivery.
General methods for efficient delivery of biologically active proteins into intact cells, in vitro and in vivo include chemical addition of a lipopeptide (P. Hoffmann et al., 1988) or a basic polymer such as polylysine or polyarginine etc. (W-C. Chen et al., 1978)
Folic acid has been used as a transport moiety (C. P. Leamon and Low, 1991). However, these methods have not proved to be highly reliable or generally useful.
Recently to introduce macromolecules such as a protein into a cell interior, gene therapy becomes in the limelight but this have also problems in that targeting is incorrect. As a alternative, research on protein transduction or protein therapy is actively progressed.
Protein transduction domain (PTD) was first reported that purified human immunodeficiency virus type-1 (“HIV”) TAT protein is taken up from the surrounding medium by adding it to human cells growing in culture medium (Green et al., 1988, Frankel et al., 1988). After this report, drosophila homeotic transcription factor, antennapedia (Antp) (Joliot et al., 1991) and herpes simplex virus-1 DNA-binding protein, VP22 (Elliot et al 1997) were also identified.
In comparison of amino acid sequences of the PTDs such as TAT, Antp and VP22 etc., basic amino acids such as arginine and lysine exist for the most part (TABLE 1) and this sequence potentiates easy approach near to the negatively charged phospholipid bilayer and penetration into the cell interior. Protein sequences having penetrating activity were named as protein transduction domains (PTDs).
TABLE 1PTDAmino Acid SequencesSEQ ID NO:HIV-1 TATTyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg82 HSV VP22Asp-Ala-Ala-Thr-Ala-Thr-Arg-Gly-Arg-Ser-Ala-Ala-83Ser-Arg-Pro-Thr-Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-Ser-Arg-Pro-Arg-Arg-Pro-Val-Glu AntpArg-Gln-Iso-Lys-Iso-Trp-Phe-Gln-Asn-Arg-Arg-Met-84Lys-Trp-Lys-Lys
In particular, recombinant expression vector was developed by using a peptide containing 11 amino acids of TAT 47-57 and TAT fusion proteins were prepared by linking the TAT peptide to other peptides or proteins and so introduction of full-length protein into intracellular compartment became possible without the limitation of size or function (Nagahara et al., 1988).
As PTDs can be linked with other peptide or proteins to form fusion protein and then be transduced into cell interior, there are many attempts to transduce therapeutic drug, peptide, protein etc. into cell interior using PTDs.
Recently, it has been known for PTDs which do not contain lots of basic amino acid residues. Also, it has been reported that PTDs penetrate phosphoelipid bilayer of cell membrane by helix conformation.
TCTP (translationally controlled tumor protein) is a protein known as IgE-dependent histamine-releasing factor (HRF) as reported by MacDonald et al. (1995). TCTP had been known as tumor-specific protein until 1980′ and the synthesis thereof was assumed to be related to proliferative stage of tumor. TCTP was reported as a tumor protein of 21 kDa, p21 in mouse erythroleukemia cell line (Chitpatima et al., 1988). Also, it was revealed that p23, relating to cell growth in Ehrlich ascites tumor is the same as TCTP/HRF (Bohm et al, 1989).
TCTP is frequently found in tumor cell, particularly growing vigorously, and exists in cytoplasm. It is a known protein consisting of 172 amino acids (NCBI accession #P13693 (Homo sapiens)) and shows high homology between species. 45 amino acids at its C-terminal form basic domain. Because such domain has about 46% homology with MAP-1B, microtubule-associated protein, it was also assumed that HRF is a microtubule-associated protein. Gachet, et al. (1997) observed that HRF is distributed consistently along with the cytoskeletal network to some extent using confocal microscope, which suggests that HRF binds to the cytoskeleton.
TCTP expression is characterized by that mRNA is maintained in regular level, but in case that exterior stimulus such as serum exists, it is transformed to polysome to be translated. According to the characteristic, it was named as ‘Translationally Controlled Tumor Protein (TCTP)’ (Thomas et al., 1981; Thomas and Thomas., 1986). It was also reported that TCTP mRNA is suppressed during translation, but when it receives cell division signal, it is activated and translated to protein (Thomas and Thomas, 1986).
TCTP/HRF is considered as a histamine releasing material interacting with basophil or mast cell and related to allergic inflammatory response.
MacDonald, et al. (1995) also found that though HRF is an intracellular protein, HRF in the outside of cells stimulates IgE-sensitized basophils to release histamine (Schroeder, et al., 1996). Schroeder, et al. (1997) observed that HRF can augment the anti-IgE-induced histamine release from all basophils, regardless of the IgE absence, and thus suggested that HRF exerts its function by binding to cell membrane receptors, not by binding with IgE.
The present inventors have previously reported that TCTP/HRF is interacted with third cytoplasmic domain (CD3) of subunit of (Na,K)ATPase thereby suppressing the activity of (Na,K)ATPase (as shown in KR Patent Application No. 10-2001-0027896) (Jung et al., 2004).
At the same time the present inventors reports that TCTP/HRF can pass through cell membrane. Since the amino acid sequence of TCTP/HRF has no part consisting of plenty of basic amino acids, arginine or lysine, which is a characteristic of representative PTDs, and no similar amino acid sequences to those of other PTDs, the present inventors considered TCTP has a domain which is different to other known PTDs in aspect of the protein structures.
In whole structure of TCTP, N- and C-terminus get loose and exposed and middle part forms a spherical shape.
In prediction of third structure, there are three helixes, wherein first helix (H1) is very short, second (H2) and third helix (H3) are exposed to outside. By H2 and H3 structure of TCTP in Schizosaccharomyces pombe, basic amino acids are distributed to outside of helix (Thaw et al., 2001) and so H2 and H3 were predicted to be related to protein transduction activity. However, by a test result, this helix part had nothing to do with translocation.
Therefore if we identify amino acid sequences with protein transduction function in TCTP/HRF, it may be possible to find new types of PTD, as well as to make a new drug delivery system though a novel vector development using these.
The present inventors made a constant effort for looking for PTD in TCTP and, as a result, isolated protein transduction domain composed of very different amino acids in comparison with well-known PTDs. On the basis of this result, the present inventors have established the present invention by confirming that this domain shows remarkably high cell penetrating activity than well-known PTDs.